DESCRIPTION: We have previously developed methodology to engineer the incorporation of a potent, foreign affinity tag (3xFLAG) into the HIV-1 proteome. As these engineered viruses are generated through a process based on viability, the tagged and essential viral protein must remain functional, likely to undergo the same interactions encountered by the wild type virus. Furthermore, because the foreign tag is stably accommodated over several cycles of viral growth, high-level expression and purification of a specific tagged viral protein can be obtained through biological amplification of the recombinant virus. In conjunction with cryogenic preservation of transient viral-host interactions and differential isotopic mass spectrometry techniques that can differentiate specific from nonspecific protein interactions, unequivocal identification of host complexes that specifically interact with the viral machinery during progressive infection has been made possible. In this proposal, we focus on another powerful attribute of our epitope-tagged viruses-the potential for selective and quantitative purification of maturational intermediates of a given vira protein directly from infected cells. Our focus here is the viral envelope protein (Env), arguably the most structurally and biochemically diversified protein of the virus and as the only solvent exposed protein of the virion, the most relevant target for vaccine development. Remarkably and unpredictably, our prototype Env 3xFLAG-tagged virus, Env-3xF, has provided us with the ability to preferentially recover and purify immature Env derivative(s) from a defined step-soon after its translation in the endoplasmic reticulum-at a point when the protein is neither proteolytically processed nor fully glycosylated but exists as a high-mannose, trimerized derivative of gp160. Here, we propose a model whereby neutralization-specific epitopes may be exposed at steps along the Env maturation pathway, when extensive glycosylation is limited and chaperone-mediated folding of structural intermediates of the glycoprotein is in progress. In addition to our prototype Env-3xF virus, we propose to construct and select an additional set of viruses derived from our original TCLA proviral clone as well as a transmitted viral founder clone (CH077) targeting different functional domains of gp120 or gp41 for 3xFLAG tag incorporation. Selective immunopurification of native maturational intermediates of Env may enrich for those rudimentary molecules in which rare, neutralization-sensitive epitopes, masked in the fully mature protein, are exposed and in a proper context to elicit a relevant immunological response. Such a panel of replication competent viruses may offer a wide spectrum of unique biochemical and/or immunological properties. Thus, the modified viruses will be expanded by biological amplification, the Env protein immunopurified and eluted under native conditions and then evaluated for the generation of broadly neutralizing antibodies in rabbits.